Adenovirus infection and transfection will be used to investigate the mechanisms underlying both homologous and non-homologous recombinational repair in mammalian cells, an area of great current interest. Double- strand breaks (DSBs) will be created in adenovirus genomes in vivo, using yeast HO endonuclease expressed from an adenoviral vector. Initial experiments will be conducted to determine if DSB-repair can occur in adenovirus-infected cells. If it can, repair of DSBs in both non- homologous and homologous pathways will be examined, using co-infection with viruses containing either appropriately located HO sites or the HO gene. The creation of DSBs in vivo also allows a direct measure of the ability of variant cells to repair such breaks, and experiments will be performed with a set of X-ray sensitive Chinese hamster cell lines. Work from others has shown that the adenovirus E4 products, ORF3p and ORF6p, prevent viral DNA concatemer formation late in infection. Experiments are proposed to distinguish between the hypothesis that these products interfere with the normal cellular response to DSBs, or alternatively that they are necessary for faithful DNA replication. Direct evidence will be sought that ORF3p and/or ORF3p and/or ORF6p inhibit end- joining between adenovirus DNA molecules introduced into cells by transfection, or following the creation of a DSB in vivo using yeast HO endonuclease. Cell lines that express either or both of the products will be tested for the inhibition of end-joining. The alternative hypothesis, that ORF3p and ORF6p are required for normal DNA replication, will be tested by examining the dependence of concatemer formation on prior DNA synthesis. To begin to analyze the mechanism of action of the E4 products, the yeast two-hybrid system, and alternative immunological and biochemical methods, will be used to try to identify cellular or viral proteins that interact with ORF3p and/or ORF6p. Positive clones from the two hybrid screen will be sequenced, and preliminary functional characterization of their products will be performed. First, appropriate nuclear extracts will be made from cells infected with wild type or E4 mutants, to see if they differ in end-joining activity, or DNA replication ability. Second, ORF3p and/or ORF6p will be added back to the extracts, to see if either product has a direct effect on biochemical activity. Third, if interacting cellular proteins can be identified and isolated, they will be added to the extracts to determine their role in conjunction with ORF3p and/or ORF6p.